关键词: 熊蜂短膜虫, PCR技术, 内转录间隔区, 熊蜂, 检疫

Abstract:  【Aim】 As a highly effective pollinator for crops, bumblebee has been widely used in greenhouse and acquired good economical and ecological benefits in foreign countries in recent years. Now many domestic enterprises have imported bumblebees and used them in facility agriculture. Crithidia bombi is one of the most important parasites of bumblebees. Once introduced with importation, it can pose significant risks to domestic bumblebees. So a rapid detection technique for C. bombi should be established urgently. 【Methods】 A PCR detection method including a pair of primers, Cri-F and Cri-R, based on the ITS sequence was established for detection of C. bombi in bumblebee bodies. After preliminarily testing the PCR result, we further optimized the reaction conditions of PCR, including the annealing temperature, primer concentration, cycle number and so on. Meanwhile, the sensitivity, specificity and stability of the PCR method were evaluated as well. 【Results】 The results showed that the PCR method with specific primers from conserved regions of ITS was feasible for detection of C. bombi. The optimized reaction conditions of PCR are the annealing temperature of 59℃, the primer concentration of 0.5 μmol/L, and the amplification cycle number of 35. The sensitivity of the detection method to the total DNA of bumblebee was approximately 13.24×10^-5 ng/μL, with a good specificity and stability. When the PCR method was applied in detection of C. bombi, the detection could be finished within 4 h, showing a good applicability. 【Conclusion】 The rapid detection method developed in this study can be applied in detection of C. bombi, which is important for epidemic surveillance of this parasite and the inspection and quarantine of imported bumblebees.

Key words: Crithidia bombi, PCR technique, internal transcribed space (ITS), bumblebees, quarantine